Functional OmpA of Salmonella Typhimurium provides protection from lysosomal degradation and inhibits autophagic processes in macrophages.

Our previous study showed that OmpA-deficient Salmonella Typhimurium (STM) failed to retain LAMP-1, quit Salmonella-containing vacuole (SCV) and escaped to the host cytosol. Here we show that the cytosolic population of STM ΔompA sequestered autophagic markers, syntaxin17 and LC3B in a sseL-dependent manner and initiated lysosomal fusion. Moreover, inhibition of autophagy using bafilomycinA1 restored its intracellular proliferation. Ectopic overexpression of OmpA in STM ΔsifA restored its vacuolar niche and increased interaction of LAMP-1, suggesting a sifA-independent role of OmpA in maintaining an intact SCV. The OmpA extracellular loops impaired the LAMP-1 recruitment to SCV and caused bacterial release into the cytosol of macrophages, but unlike STM ΔompA, they retained their outer membrane stability and didn't activate the lysosomal degradation pathway aiding in their intra-macrophage survival. Finally, OmpA extracellular loop mutations protected the cytosolic STM ΔsifA from the lysosomal surveillance, revealing a unique OmpA-dependent strategy of STM for its intracellular survival.

Clustering of Polymorphic Membrane Protein E Clade in Chlamydia trachomatis Lineages from Men Who Have Sex with Men.

Several Chlamydia trachomatis lineages identified through outer membrane protein A genotyping or multilocus sequence typing have been circulating worldwide among men who have sex with men. In a study in Tokyo, Japan, we demonstrate that such lineages commonly belong to a specific polymorphic membrane protein E clade across genotypes.

Oral administration of recombinant outer membrane protein A-based nanovaccine affords protection against Aeromonas hydrophila in zebrafish.

Aeromonas hydrophila, an opportunistic warm water pathogen, has always been a threat to aquaculture, leading to substantial economic losses. Vaccination of the cultured fish would effectively prevent Aeromoniasis, and recent advancements in nanotechnology show promise for efficacious vaccines. Oral delivery would be the most practical and convenient method of vaccine delivery in a grow-out pond. This study studied the immunogenicity and protective efficacy of a nanoparticle-loaded outer membrane protein A from A. hydrophila in the zebrafish model. The protein was over-expressed, purified, and encapsulated using poly lactic-co-glycolic acid (PLGA) nanoparticles via the double emulsion method. The PLGA nanoparticles loaded with recombinant OmpA (rOmpA) exhibited a size of 295 ± 15.1 nm, an encapsulation efficiency of 72.52%, and a polydispersity index of 0.292 ± 0.07. Scanning electron microscopy confirmed the spherical and isolated nature of the PLGA-rOmpA nanoparticles. The protective efficacy in A. hydrophila-infected zebrafish after oral administration of the nanovaccine resulted in relative percentage survival of 77.7. Gene expression studies showed significant upregulation of immune genes in the vaccinated fish. The results demonstrate the usefulness of oral administration of nanovaccine-loaded rOmpA as a potential vaccine since it induced a robust immune response and conferred adequate protection against A. hydrophila in zebrafish, Danio rerio.

Facile One-Pot Three Component Synthesis, Characterization, and Molecular Docking Simulations of Novel α-Aminophosphonate Derivatives Based Pyrazole Moiety as Potential Antimicrobial Agent.

An efficient method has been developed for the synthesis of novel α-aminophosphonates (AAP) (3 a-m) through a one-pot three-component reaction of 1,3-disubstituted-1H-pyrazol-5-amine, aromatic aldehydes, and phosphite using lithium perchlorate as catalyst. All newly synthesized compounds were characterized via different spectroscopic techniques. The synthesized compounds' mode of action was investigated using molecular docking against the outer membrane protein A (OMPA) and exo-1,3-ß-glucanase, with interpreting their pharmacokinetics aspects. The results of the antimicrobial effectiveness of these compounds revealed a broad spectrum of their biocidal activity and this in-vitro study was in line with the in- silico results. Additionally, it has been demonstrated that these compounds exhibited a minimum inhibitory concentration (MIC) with significant activity at low concentrations (7.5-30.0 mg/mL). Further, the radical scavenging (DPPH* ) activity of the synthesized compounds fluctuated, with compounds 3 h, 3 a, and 3 f showing the highest antioxidant activity. Overall, the formulated compounds can be employed as antimicrobial and antioxidant agents in medical applications.

The OmpA of commensal Escherichia coli of CRC patients affects apoptosis of the HCT116 colon cancer cell line.

BACKGROUND: Colorectal cancer ranks third globally among all types of cancers. Dysbiosis of the gut microbiota of people with CRC is one of the effective agents in the tumorigenesis and metastasis in this type of cancer. The population of Escherichia coli strains, a component of gut microbiota, is increased in the gut of people with CRC compared with healthy people. So, E.coli strains isolated from these patients may have a role in tumorigenesis. Because the most isolated strains belong to the B2 phylogenuetic group, there seems to be a linkage between the bacterium components and malignancy. MATERIAL AND METHODS: In this study, the proteomic comparison between isolated Ecoli from CRC patients and healthy people was assayed. The isolated spot was studied by Two-dimensional gel electrophoresis (2DE) and Liquid chromatography-mass spectrometry (LC-MS). The results showed that the expression of Outer membrane protein A (OmpA) protein increased in the commensal E.coli B2 phylogenetic group isolated from CRC patients. Additionally, we analyzed the effect of the OmpA protein on the expression of the four genes related to apoptosis in the HCT116 colon cancer cell line. RESULTS: This study identified that OmpA protein was overexpressed in the commensal E.coli B2 phylogenetic group isolated from CRC patients compared to the E.coli from the control group. This protein significantly decreased the expression of Bax and Bak, pro-apoptotic genes, as well as the expression of P53 in the HCT116 Cell Line, P < 0.0001. LC-MS and protein bioinformatics results confirmed that this protein is outer membrane protein A, which can bind to nucleic acid and some of the organelle proteins on the eukaryotic cell surface. CONCLUSIONS: According to our invitro and insilico investigations, OmpA of gut E.coli strains that belong to the B2 phylogenetic group can affect the eukaryotic cell cycle.

Trigger factor is a bona fide secretory pathway chaperone that interacts with SecB and the translocase.

Bacterial secretory preproteins are translocated across the inner membrane post-translationally by the SecYEG-SecA translocase. Mature domain features and signal peptides maintain preproteins in kinetically trapped, largely soluble, folding intermediates. Some aggregation-prone preproteins require chaperones, like trigger factor (TF) and SecB, for solubility and/or targeting. TF antagonizes the contribution of SecB to secretion by an unknown molecular mechanism. We reconstituted this interaction in vitro and studied targeting and secretion of the model preprotein pro-OmpA. TF and SecB display distinct, unsuspected roles in secretion. Tightly associating TF:pro-OmpA targets the translocase at SecA, but TF prevents pro-OmpA secretion. In solution, SecB binds TF:pro-OmpA with high affinity. At the membrane, when bound to the SecA C-tail, SecB increases TF and TF:pro-OmpA affinities for the translocase and allows pro-OmpA to resume translocation. Our data reveal that TF, a main cytoplasmic folding pathway chaperone, is also a bona fide post-translational secretory chaperone that directly interacts with both SecB and the translocase to mediate regulated protein secretion. Thus, TF links the cytoplasmic folding and secretion chaperone networks.

Enhancement of Acinetobacter baumannii biofilm growth by cephem antibiotics via enrichment of protein and extracellular DNA in the biofilm matrices.

AIMS: The aims were to determine the effects of subinhibitory concentrations of eight cephem and carbapenem antibiotics on the biofilm formation of Acinetobacter baumannii cells and examine their effects on pre-established biofilms. METHODS AND RESULTS: Effects of antibiotics on biofilm formation were assayed using microtitre plates with polystyrene peg-lids. Cefmetazole, ceftriaxone, ceftazidime and cefpirome increased the biomass of pre-established biofilms on pegs in the range of their sub-minimum inhibitory concentrations (MICs), whereas none increased biofilm formation by planktonic cells. Carbapenems had a negative effect. The constituents of antibiotic-induced biofilms were analysed. Ceftriaxone or ceftazidime treatment markedly increased the matrix constituent amounts in the biofilms (carbohydrate, 2.7-fold; protein, 8.9-12.7-fold; lipid, 3.3-3.6-fold; DNA, 9.1-12.2-fold; outer membrane vesicles, 2.7-3.8-fold and viable cells, 6.8-10.1-fold). The antibiotic-enhanced biofilms had increased outer membrane protein A and were resistant to the anti-biofilm effect of azithromycin. CONCLUSIONS: Some cephems increased the biomass of pre-established biofilms in the ranges of their sub-MICs. The antibiotic-enhanced biofilms possessed more virulent characteristics than normal biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: Incomplete administration of certain cephems following biofilm-related Ac. baumannii infections could adversely cause exacerbated and chronic clinical results.

Evaluation the reactivity of a peptide-based monoclonal antibody derived from OmpA with drug resistant pulsotypes of Acinetobacter baumannii as a potential therapeutic approach.

BACKGROUND: Acinetobacter baumannii is an opportunistic and antibiotic-resistant pathogen that predominantly causes nosocomial infections. There is urgent need for development nonantibiotic-based treatment strategies. We developed a novel monoclonal antibody (mAb) against a peptide of conserved outer membrane protein A (OmpA) and evaluated its reactivity with different pulsotypes of A. baumannii. METHODS: Peptide derived from A. baumannii OmpA was conjugated to keyhole limpet hemocyanin and injected into BALB/c mice. Splenocytes of immunized mice were fused with SP2/0 myeloma cells followed by selection of antibody-producing hybridoma cells. After screening of different hybridoma colonies by ELISA, one monoclone was selected as 3F10-C9 and the antibody was tested for reaction with five different Acinetobacter pulsotypes that were resistant to carbapenem antibiotics. The affinity constant was measured by ELISA. The ELISA, western blotting, indirect immunofluorescence (IFA), and in vitro opsonophagocytosis assays were used to evaluate the reactivity of generated mAb. RESULTS: The anti-OmpA antibody reacted with the immunizing peptide and had a high affinity (1.94 × 10-9 M) for its antigen in the ELISA. Specific binding of mAb to OmpA was confirmed in Western blot. IFA assays revealed that mAb recognized specific OmpA on the pulsotypes. Opsonophagocytosis assays showed that the mAb increased the bactericidal activity of macrophage cells. The antibody function was higher in the presence of serum complement. CONCLUSIONS: The peptide-based mAb demonstrated optimal performance in laboratory experiments which may be appropriate in investigation on OmpA in Acinetobacter pathogenesis and development of passive immunization as a novel therapeutic approach.

Outer membrane protein A inhibits the degradation of caspase-1 to regulate NLRP3 inflammasome activation and exacerbate the Acinetobacter baumannii pulmonary inflammation.

Acinetobacter baumannii (A. baumannii), one of the major pathogens that causes severe nosocomial infections, is characterised by a high prevalence of drug resistance. It has been reported that A. baumannii triggers the NOD-like receptor 3 (NLRP3) inflammasome, but the role of its virulence-related outer membrane protein A (ompA) remains unclear. Therefore, this study aimed to explore the effects of ompA on the NLRP3 inflammasome and its underlying molecular mechanisms. Results showed that ompA enhanced inflammatory damage, which was reduced as a result of knockout of the ompA gene. Additionally, ompA-stimulated expression of NLRP3 inflammasome was significantly blocked by silencing caspase-1, but activation of NLRP3 inflammasome was not altered after silencing ASC; this indicated that ompA was dependent on the caspase-1 pathway to activate the inflammatory response. Simultaneously, the wild-type (WT) strains triggered NLRP3 inflammasome after inhibition of caspase-1 degradation by proteasome inhibitor MG-132, aggravating tissue damage. These findings indicated that ompA may be dependent on the caspase-1 pathway to enhance inflammation and exacerbate tissue damage. Taken together, these results confirmed a novel capsase-1-modulated mechanism underpinning ompA activity, which further reveals the NLRP3 inflammasome pathway as a potential immunomodulatory target against A. baumannii infections.

Immunization of a novel outer membrane protein from Aeromonas hydrophila simultaneously resisting A. hydrophila and Edwardsiella anguillarum infection in European eels (Angullia angullia).

In cultivated European eels, Aeromonas hydrophila, Edwardsiella anguillarum and Vibrio vulnificus are three important bacterial pathogens. In this study, an expressed recombinant Outer membrane proteinⅡ (rOmpⅡ) from A. hydrophila was intraperitoneally injected into European eels (Angullia angullia). All examined eels were equally divided into three groups. One group was injected with PBS only (PBS group), one group was injected with 1:1 mixture of PBS and Freund's incomplete adjuvant (PBS + F, adjuvant group), and the third group was injected with 1:1 mixture of 1 mg mL-1 rOmpⅡ and Freund's incomplete adjuvant (rOmpⅡ+F, OmpⅡ group). The immunogenicity of OmpⅡ was studied by detecting the expression of 4 immune-related genes, stimulation index (SI) of the whole blood cell, serum antibody titer, lysozyme and Superoxide Dismutase (SOD) activity, and relative percent of survival (RPS) rate. The results showed that gene expression of MHC-Ⅱ, LysC, SOD and IgM in the OmpⅡ group significantly increased in liver, spleen, kidney and intestine. At 28 days post the immunization (dpi), the SI of whole blood cells in the OmpⅡ group increased significantly; at 14, 21, 28 and 42 dpi, the serum antibody titers against A. hydrophila and E. anguillarum in the OmpⅡ group were significantly higher than that of the PBS and the adjuvant group; the SOD in the OmpⅡ group was found increased significantly in liver, kidney, mucus and serum. On the 28 dpi, eels were challenged by A. hydrophila, E. anguillarum and V. vulnificus for cross protection study. The results showed that the RPS of the OmpⅡ group were 83.33%, 55.56% and 33.33% respectively. These results showed that the expressed OmpⅡ from A. hydrophila significantly improve the immune function of Europena eels and their resistance to the infection of A. hydrophila and E. anguillarum simultaneously.

Assessment of immunization regimens of duck Riemerella anatipestifer vaccines.

AIMS: Riemerella anatipestifer infections of goslings and ducklings can result in high mortality. Since there are at least 21 serotypes of R. anatipestifer, cross-protection is an important goal for vaccine development. METHODS AND RESULTS: In this study, we evaluated the immunostimulatory effect of different immunization regimens - the traditional inactivated vaccine vs prime-boost regimens using DNA and protein subunit vaccines (DNA+subunit, subunit+subunit, subunit+inactivated and DNA+DNA). Results showed that, when compared to the inactivated vaccine, prime-boost regimens induced higher and up to 16-week longer lasting levels of antibody responses, significantly elevated the percentage of the cytotoxic CD8+ T cell and higher expression levels of IFN-γ, IL-6 and IL-12 mRNAs. Furthermore, as an indication of cross-protection, sera from prime-boost regimens were able to recognize lysates of R. anatipestifer serotypes 1, 2 and 6. CONCLUSIONS: Prime-boost regimens especially DNA-prime and protein-boost, induce strong long-term immune response and may prove protective for breeder ducks requiring long-term protection. SIGNIFICANCE AND IMPACT OF THE STUDY: It is worth mentioning that the subunit+inactivated regimen group also elicited strong immune response. The cost of this regimen may only be half of the other prime-boost regimens, making this subunit + inactivated combination an attractive option.

Evaluation of the protective immunity of Riemerella anatipestifer OmpA.

Riemerella anatipestifer is responsible for an economically important disease of commercially raised ducks. No or only few cross-protection was observed between different serotypes of R. anatipestifer strains, and so far no protective antigen in this bacterium has been identified. OmpA is a predominant immunogenic protein of R. anatipestifer, and within the 1467 bp ompA ORF (ompA1467), there is another 1164 bp ORF (ompA1164) with the same C-terminal. In this study, our results showed that the full sequence of ompA1467 from some R. anatipestifer strains with different serotypes shared the same amino acid sequence. Animal experiments showed that the soluble recombinant protein rOmpA1164, but not rOmpA1467, could provide partial protective immunity against challenge. Moreover, there was no significant difference in protective immunity between ducklings immunized with Th4△ompA bacterin and those immunized with Th4 bacterin. In addition, OmpA1467 was the main existing form of OmpA in R. anatipestifer cells by gel electrophoresis and western blot analyses. The results suggested that OmpA1467 was not a protective antigen of R. anatipestifer, and antibodies against proteins other than OmpA play a critical role in the process of anti-R. anatipestifer infection.

In vivo antibacterial activity of Zataria multiflora Boiss extract and its components, carvacrol, and thymol, against colistin-resistant Acinetobacter baumannii in a pneumonic BALB/c mouse model.

BACKGROUND: Acinetobacter baumannii has emerged as a major cause of nosocomial infections. Various resistance mechanisms of A. baumannii against antibiotics have transformed it into a successful nosocomial pathogen. Because of the limited number of available antibiotics, we used a medicinal plant with an antibacterial effect. Zataria multiflora Boiss (ZMB) extract and its components were used for the treatment of pneumonic mice infected with A. baumannii. The biological effects of this extract and the regulation of the outer membrane protein A (ompA) gene were used in a mouse model. METHODS: A pneumonic mouse model was prepared using clinical and standard strains (1.5 × 108 colony-forming units/mL) of A. baumannii. BALB/c mice groups were treated with a ZMB extract, carvacrol, thymol, and sensitive antibiotics. The lung tissues of the treated mice were cultured for 5 days and each day, bacterial clearance and the ompA gene expression were assessed by quantitative real-time polymerase chain reaction. RESULTS: In the lung tissue culture of pneumonic mice infected with standard or clinical isolate, no colony was detected when treated with the ZMB extract after 2 and 3 days (P < 0.01), respectively. In the carvacrol-treated group, bacterial clearance was seen at day 4 and day 5 (P < 0.05). Bacterial clearance was seen 5 days after treatment with thymol and imipenem and 6 days after ampicillin/sulbactam treatment. The regulation of ompA gene was significantly decreased in this order: ZMB extract, carvacrol, thymol, imipenem, and ampicillin/sulbactam. DISCUSSION: The ZMB extract had a potent bactericidal effect against A. baumannii that could downregulate the ompA gene. ZBM extract and carvacrol could be novel therapeutic agents for antibiotic-resistant A. baumannii.

Acinetobacter baumannii outer membrane protein A induces HeLa cell autophagy via MAPK/JNK signaling pathway.

Autophagy is an evolutionary conserved self-balancing process that plays an important role in maintaining cellular homeostasis via the clearance of damaged organelles and misfolded proteins. Infection-triggered autophagy specifically inhibits the invasion of intracellular bacterial replication and hence protects the cells from microbial infections. It has been reported that Acinetobacter baumannii trigger cell autophagy. However, the role of its virulence protein OmpA remains unclear. Therefore, this study aimed to explore the effects of Acinetobacter baumannii OmpA on cell autophagy and its underlying molecular mechanisms. The results showed that OmpA induced autophagy in HeLa and RAW264.7 cells, increased LC3BII expression, and hindered p62 degradation. Moreover, OmpA triggered incomplete autophagy by interfering the fusion of autophagosomes with lysosomes. Besides, OmpA activated MAPK/JNK signaling pathway and enhanced the phosphorylation levels of JNK, p38, and ERK, c-Jun. Inhibition of JNK signaling pathway suppressed OmpA-induced autophagy in HeLa cells. Ab wild-type strains carrying OmpA triggered incomplete autophagy and resulted in a large number of IL-1ß production. Ab-△OmpA strain (OmpA gene mutation) restored autophagic flux and reduced the accumulation of p62 and the release of IL-1ß in HeLa cells. Rapamycin activated autophagy to inhibit OmpA-induced IL-1ß secretion and protect HeLa cells from inflammatory damage. Collectively, these results suggest that OmpA can induce autophagy in HeLa cells through MAPK/JNK signaling pathway. Pre-treatment with Rapamycin activates autophagy and protects against cell death.

Antigenic epitopes on the outer membrane protein A of Escherichia coli identified with single-chain variable fragment (scFv) antibodies.

Bacterial meningitis is a severe disease that is fatal to one-third of patients. The major cause of meningitis in neonates is Escherichia coli (E. coli) K1. This bacterium synthesizes an outer membrane protein A (OmpA) that is responsible for the adhesion to (and invasion of) endothelial cells. Thus, the OmpA protein represents a potential target for developing diagnostic and therapeutic agents for meningitis. In this study, we expressed recombinant OmpA proteins with various molecular weights in E. coli. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to check the molecular size of OmpA's full length (FL) and truncated proteins. OmpA-FL protein was purified for immunizing chickens to produce immunoglobulin yolk (IgY) antibodies. We applied phage display technology to construct antibody libraries (OmpA-FL scFv-S 1.1 × 107 and OmpA-FL scFv-L 5.01 × 106) to select specific anti-OmpA-FL scFv antibodies; these were characterized by their binding ability to recombinant or endogenous OmpA using ELISA, immunofluorescent staining, and confirmed with immunoblotting. We found 12 monoclonal antibodies that react to OmpA fragments; seven scFvs recognize fragments spanning amino acid (aa) residues 1-346, aa 1-287, aa 1-167, and aa 60-192, while five scFvs recognize fragments spanning aa 1-346 and aa 1-287 only. Two fragments (aa 246-346 and aa 287-346) were not recognized with any of the 12 scFvs. Together, the data suggest three antigenic epitopes (60 aa-160 aa, 161 aa-167 aa, 193 aa-245 aa) recognized by monoclonal antibodies. These scFv antibodies show strong reactivity against OmpA proteins. We believe that antibodies show promising diagnostic agents for E. coli K1 meningitis.

Differential effects of Th17 cytokines during the response of neutrophils to Burkholderia cenocepacia outer membrane protein A.

T helper 17 cells are involved in the immunopathology of cystic fibrosis. They play a key role in recruitment of neutrophils, which is the first line of defence against bacteria. Additionally, Burkholderia cenocepacia outer membrane protein A (OmpA) BCAL2958 is considered a potential protective epitope for vaccine development. The present study aimed to investigate the neutrophil response to OmpA in the presence of Th17 cytokines, IL-17 and IL-22 at different times of activation. Neutrophils were isolated from whole blood of healthy volunteers and activated with OmpA in the presence of IL-17, IL-22 or both cytokines together. Supernatant was collected after 1 h, 2 h, 4 h, 8 h, and 12 h. Neutrophil activation was assessed by measuring MPO, TNF-α, elastase, hydrogen peroxide, catalase and NO. The results revealed that the combination of IL-17 and IL-22 cytokines induced the release of NE, catalase, H2O2 and TNF-α from neutrophils activated with Burkholderia OmpA at late stages of activation. However, IL-22 alone or IL-17 alone decreased the myeloperoxidase (MPO), catalase and NE levels at early stages of neutrophil activation. The presence of IL-17 alone led to a significant increase in TNF-α level after 1 h and 12 h. However, the presence of IL-22 alone led to a significant increase in TNF-α level after only 1 h but a significant decrease after 8 h of activation was observed as compared to OmpA stimulated neutrophils. In conclusion, Th17 cytokines IL-17 and IL-22, have differential effects during the neutrophil response to Burkholderia OmpA.

A Simplified Derivative of Human Defensin 5 with Potent and Efficient Activity against Multidrug-Resistant Acinetobacter baumannii.

The increasing incidence of multidrug-resistant Acinetobacter baumannii (MDRAb) infections worldwide has necessitated the development of novel antibiotics. Human defensin 5 (HD5) is an endogenous peptide with a complex architecture and antibacterial activity against MDRAb In the present study, we attempted to simplify the structure of HD5 by removing disulfide bonds. We found that the Cys2-4 bond was most indispensable for HD5 to inactivate MDRAb, although the antibacterial activity of the derivative was significantly attenuated. We then replaced the noncationic and nonhydrophobic residues with electropositive Arg to increase the antibacterial activity of HD5 derivative that contains a Cys2-4 bond, obtaining another derivative termed HD5d5. The in vitro antibacterial assay and irradiation-wound-infection animal experiment both showed that HD5d5 was much more effective than HD5 at eliminating MDRAb Further investigations revealed that HD5d5 efficiently bound to outer membrane lipid A and penetrated membranes, leading to bacterial collapse and peptide translocation. Compared to HD5, more HD5d5 molecules were located in the cytoplasm of MDRAb, and HD5d5 was more efficient at reducing the activities of superoxide dismutase and catalase, causing the accumulation of reactive oxygen species that are detrimental to microbes. In addition, HD5 failed to suppress the pathogenic outer membrane protein A of Acinetobacter baumannii (AbOmpA) at concentrations up to 50 µg/ml, whereas HD5d5 strongly bound to AbOmpA and exhibited a dramatic toxin-neutralizing ability, thus expanding the repertoire of drugs that is available to treat MDRAb infections.

The Capsular Polysaccharide of Acinetobacter baumannii Is an Obstacle for Therapeutic Passive Immunization Strategies.

Acinetobacter baumannii has become an important concern for human health due to rapid development and wide spread of antimicrobial-resistant strains and high mortality associated with the infection. Passive immunizations with antisera targeting outer membrane proteins (OMPs) have shown encouraging results in protecting mice from A. baumannii infection, but monoclonal anti-OMP antibodies have not been developed, and their potential therapeutic properties have not been explored. The goal of this report is to evaluate the antibacterial activity of monoclonal antibodies (MAbs) targeting outer membrane protein A (OmpA) of A. baumannii Five anti-OmpA MAbs were developed using hybridoma technology and showed strong binding to strain ATCC 19606. However, low antibody binding was observed when they were tested against six clinical isolates, which included extensively drug-resistant strains. In contrast, high binding to an isogenic K1 capsule-negative mutant (AB307.30) was shown, suggesting that capsular polysaccharide mediated the inhibition of MAb binding to OmpA. Anti-OmpA MAbs increased the macrophage-mediated bactericidal activity of AB307.30 but failed to increase phagocytic killing of capsule-positive strains. Capsular polysaccharide was also protective against complement-mediated bactericidal activity in human ascites in the presence and absence of opsonization. Lastly, passive immunization with anti-OmpA MAbs did not confer protection against challenge with AB307-0294, the encapsulated parent strain of AB307.30, in a mouse sepsis infection model. These results reveal the important role of capsule polysaccharide in shielding OmpA and thereby inhibiting anti-OmpA MAb binding to clinical isolates. This property of capsule hindered the therapeutic utility of anti-OmpA MAbs, and it may apply to other conserved epitopes in A. baumannii.

Outer membrane protein A: An immunogenic protein induces highly protective efficacy against Vibrio ichthyoenteri.

Vibrio ichthyoenteri was an important causative agent of bacterial enteritis in flounder (Paralichthys olivaceus). Outer membrane protein A (OmpA) of Gram-negative pathogen was a major cell surface antigen. In the present study, OmpA of V. ichthyoenteri was recombinantly expressed in Escherichia coli, and the immunogenicity of OmpA was identified by western blotting using flounder anti-rOmpA and anti-V. ichthyoenteri antibodies. The vaccine potential of rOmpA was tested in a flounder model, and a high relative percentage of survival rate was obtained with 73.1% after challenge with V. ichthyoenteri. Meanwhile, the immune response of flounder induced by rOmpA was also investigated, and the results showed that the sIg + lymphocytes in blood, spleen, and pronephros significantly proliferated, and the peak levels occurred at week 4 after immunization. Moreover, rOmpA could induce higher levels of specific serum antibodies than the control group after immunization, and the peak level occurred at week 5 after immunization. Meanwhile, qRT-PCR analysis showed that the expressions of CD4-1, CD8α, IL-1ß, IFN-γ, MHCIα and MHCIIα genes were significantly up-regulated after immunization with rOmpA. Taking together, these results demonstrated that rOmpA could evoke highly protective effects against V. ichthyoenteri challenge and induce strong immune response of flounder, which indicated that OmpA was a promising vaccine candidate.

Immunization with r-Lactococcus lactis expressing outer membrane protein A of Shigella dysenteriae type-1: evaluation of oral and intranasal route of administration.

AIMS: To evaluate the comparative immunogenic potential of food grade Lactococcus lactis expressing outer membrane protein A (OmpA) of Shigella dysenteriae type-1 (SD-1) when administered either orally or intranasally. METHODS AND RESULTS: OmpA of SD-1 was cloned and expressed first in Escherichia coli and then in L. lactis. Presence of recombinant gene was confirmed by restriction enzyme digestion and immunoblot analysis. Using immobilized metal affinity chromatography, OmpA was purified from recombinant E. coliBL21 (DE3) and subcutaneously administered in BALB/c mice. Detection of OmpA-specific IgG antibodies by enzyme-linked immunosorbent assay (ELISA) confirmed the immunogenicity of OmpA. In order to establish r-L. lactis as a mucosal delivery vehicle, it was administered orally and nasally in BALB/c mice. Serum IgG and faecal IgA were assessed through ELISA to compare the relative potential of immunization routes and immunogenic potential of r-L. lactis. Immunization via the oral route proved superior to intranasal exposure. CONCLUSION: Recombinant L. lactis expressing OmpA of SD-1 was found to be immunogenic. Oral administration of r-L. lactis elicited higher systemic and mucosal immune response when compared with the nasal route. SIGNIFICANCE AND IMPACT OF THE STUDY: Using food grade recombinant L. lactis has implications in the development of a prophylactic against multidrug-resistant Shigella, which can be used as a prospective vaccine candidate. Evaluating mucosal routes of immunization demonstrated that the oral route of administration elicited better immune response against OmpA of Shigella.